Caspian Journal of Veterinary Sciences

Caspian Journal of Veterinary Sciences

Hinokitiol plus fluconazole: potent inhibitory efficacy on Candida species resistant to azole

Document Type : Original Article

Authors
Donya Nikaein 1
Hojjatollah Shokri 2
Aghil Sharifzadeh 1
Mohammadreza Mohammadian 3
Ahmad Jahanian 1
1 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
2 Department of Pathobiology, Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran.
3 Department of Animal and Poultry Health and Nutrition, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
10.22034/cjvs.2026.560426.1052
Abstract
Candida species represent the most prevalent types of yeasts affecting humans and animals. The increase in antifungal resistance has raised the issue of using these medications in tandem to improve treatment outcomes. Guided by the CLSI M27-A3 guideline, this investigation quantified the synergistic potential of hinokitiol and fluconazole against Candida albicans, C. tropicalis, C. glabrata, and C. krusei. The primary metrics for this assessment were the minimum inhibitory and fungicidal concentrations (MICs and MFCs), which were established using a broth microdilution methodology. Using data from a checkerboard microdilution test, we calculated the fractional inhibitory concentration indices (FICIs) to evaluate the interaction between hinokitiol and fluconazole. A strong synergistic interaction between hinokitiol and fluconazole was evident in every tested isolate (100%) of fluconazole-resistant C. albicans and C. glabrata. The corresponding FICI values, falling within ranges of 0.304–0.498 and 0.290–0.492, respectively, confirm this robust synergistic activity. This activity was noted against a background of universal fluconazole resistance, with hinokitiol alone showing MIC values from 0.81 µg/ml (C. glabrata) to 6.9 µg/ml (C. krusei). FICI scores spanning 0.500 to 0.750 for every C. krusei strain (100%) confirm a strictly additive pharmacological interaction between hinokitiol and fluconazole. Additionally, the FICI values for the combination of hinokitiol and fluconazole for C. tropicalis strains varied from 0.245 to 0.730, demonstrating a synergistic effect in 13 (86.7%) strains and an additive effect in 2 (13.3%) strains. This study suggests that hinokitiol may help reduce fluconazole dosages and inhibit the development of fluconazole-resistant Candida species
Keywords
Candida
Fluconazole
Hinokitiol
Synergy
Subjects
Barros, L; Dueñas, M; Alves, C; Silva, S; Henriques, M; Santos-Buelga, C and Ferreira, ICFR (2013). Antifungal activity and detailed chemical characterization of Cistus ladanifer phenolic extracts. Industr. Crops Prod., 41: 41-45.
Biswas, S; Van Dijck, P and Datta, A (2007). Environmental sensing and signal transduction pathways regulating morphopathogenic determinants of Candida albicans. Microbiol. Mol. Biol. Rev., 71: 348-376.
Carson, CF; Mee, BJ and Riley, TV (2002). Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus. Antimicrob. Agents Chemother., 46: 1914-1920.
De Castro, RD; de Souza, TMPA; Bezerra, LMD; Ferreira, GLS; de Brito Costa, EMM and Cavalcanti, AL (2015). Antifungal activity and mode of action of thymol and its synergism with nystatin against Candida species. BMC Comp. Altern. Med., 15: 417.
Desnos-Ollivier, M; Bretagne, S; Raoux, D; Hoinard, D; Dromer, F and Dannaoui, E (2008). Mutations in the fks1 gene in Candida spp. correlate with elevated caspofungin MICs. Antimicrob. Agents Chemother., 52: 3092-3098.
Domon, H; Hiyoshi, T and Maekawa, T (2019). Antibacterial activity of hinokitiol against oral pathogens. Microbiol. Immunol., 63: 213-222.
Hu, J; Shen, Y and Pang, S (2013). Application of hinokitiol potassium salt for wood preservative. J. Environ. Sci., 25: S32-S35.
Jayakumar, T; Hsu, WH and Yen, TL (2013). Hinokitiol offers neuroprotection from thromboembolic stroke in vivo. Evidence-Based Comp. Altern. Med., 2013: 840487.
Jin, J; Guo, N and Zhang, J (2010). The synergy of honokiol and fluconazole against Candida albicans. Lett. Appl. Microbiol., 51: 351-357.
Jin, X; Zhang, M and Lu, J (2021). Hinokitiol chelates intracellular iron to retard fungal growth. J. Advan. Res., 34: 65-77.
Kim, DJ; Lee, MW and Choi, JS (2017). Inhibitory activity of hinokitiol against biofilm formation in fluconazole-resistant Candida species. PLoS One, 12: e0171244.
Komaki, N; Watanabe, T and Ogasawara, A (2008). Antifungal mechanism of hinokitiol against Candida albicans. Biol. Pharm. Bull., 31: 735-737.
Ma, S; Lin, L and Peng, X (2022). Hinokitiol alters gene expression in Aspergillus fumigatus. BioRxiv. 
Makarova, NU; Pokrowsky, VV and Kravchenko, A (2003). Persistence of oropharyngeal Candida albicans strains with reduced susceptibilities to fluconazole. J. Clin. Microbiol., 41: 1833-1837.
Mori, T; Hirose, H; Hanjavanit, C and Hatai, K (2002). Antifungal activities of plant extracts against aquatic fungi. Biocontrol Sci., 7: 187-191.
Nakamura, M; Fujib. Pemmaraju, SC; Pruthi, PA; Prasad, R and Pruthi, V (2013). Candida albicans biofilm inhibition by synergistic action of terpenes and fluconazole. Indian J. Exp. Biol., 51: 1032-1037.
Sasse, C; Dunkel, N; Schäfer, T; Schneider, S; Dierolf, F; Ohlsen, K and Morschhäuser, J (2012). The stepwise acquisition of fluconazole resistance mutations causes a gradual loss of fitness in Candida albicans. Mol. Microbiol., 86: 539-556.
Sharifzadeh, A; Khosravi, A; Shokri, H and Shirzadi, H (2018). Potential effect of 2-isopropyl-5-methylphenol (thymol) alone and in combination with fluconazole against clinical isolates of Candida albicans, C. glabrata and C. krusei. J. Mycol. Méd., 28: 294-299.
Sharifzadeh, A; Khosravi, AR; Shokri, H and Tari, PS (2017). Synergistic anticandidal activity of menthol in combination with itraconazole and nystatin against clinical Candida glabrata and Candida krusei isolates. Microb. Pathog., 107: 390-396.
Sharifzadeh, A and Shokri, H (2021). In vitro synergy of eugenol on the antifungal effects of voriconazole against Candida tropicalis and Candida krusei strains isolated from the genital tract of mares. Equine Vet. J., 53: 94-101.
Shin, S and Lim, S (2004). Antifungal effects of herbal essential oils alone and in combination with ketoconazole against Trichophyton spp. J. Appl. Microbiol., 97: 1289-1296.
Shokri, H; Minooieanhaghigh, M and Sharifzadeh, A (2021). The synergistic activity of eugenol and fluconazole on the induction of necrosis and apoptosis in Candida krusei isolates of HIV+ patients with oral candidiasis. Internal Med. Today, 27: 434-449.
Sun, L; Liao, K and Wang, D (2017). Honokiol induces superoxide production by targeting mitochondrial respiratory chain complex I in Candida albicans. PLoS One, 12: e0184003.
Tobudic, S; Kratzer, C and Presterl, E (2012). Azole‐resistant Candida spp.–emerging pathogens? Mycoses, 55: 24-32.
Wang, Y; Liu, X; Chen, T; Xu, Y and Tian, S (2020). Antifungal effects of hinokitiol on development of Botrytis cinerea in vitro and in vivo. Postharvest Biol. Technol., 159: 111038.
Whaley, SG; Berkow, EL; Rybak, JM; Nishimoto, AT; Barker, KS and Rogers, PD (2017). Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front. Microbiol., 7: 2173.
Zhang, H; Chen, H; Niu, J; Wang, Y and Xie, L (2009). Role of adaptive immunity in the pathogenesis of Candida albicans keratitis. Invest. Ophthalmol. Vis. Sci., 50: 2653-2659. 
Caspian Journal of Veterinary Sciences
Volume 3, Issue 1
June 2026
Pages 23-31

  • Receive Date 19 November 2025
  • Accept Date 18 April 2026